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The Transfection of HepG2 cells with Truncated β-Catenin Coding Expression Vector

Year 2020, Volume: 7 Issue: 1, 211 - 220, 28.06.2020
https://doi.org/10.35193/bseufbd.658677

Abstract

β-catenin is an effector protein in Wnt signaling. β-catenin mutations are reported in the development of many diseases such as autism, colorectal carcinoma, developmental delay, intellectual disability, neurodegeneration, skin, hair and facial anomalies. Exon 3 deletion mediated truncations of the β-catenin associated with these diseases. Therefore understanding the functions of wild type and exon 3 deleted forms of β-catenin may provide an enhancement in the treatment of many diseases. However, to conduct controlled experiments, there could be a demand for the expression vectors that code for wild type and exon 3 deleted forms of β-catenin and originated from the same organism. Since it has long been known that HepG2 cells are heterozygous for β-catenin, in this study, it was found worthy of constructing the expression vectors from the total RNA of HepG2 cells. Then the utility of truncated β-catenin coding pcDNA3.1/CTNNB1 expression vector for upregulation of truncated β-catenin in HepG2 cells was examined. To this end, RNA was isolated from HepG2 cells, cDNA fragments were amplified by polymerase chain reaction (PCR), expression vectors were constructed then sequenced from 5’-prime regions. Following the BLAST analysis, it was concluded that both truncated and wild type β-catenin coding pcDNA3.1/CTNNB1 expression vectors were successfully cloned in E. coli cells. Interestingly, when the parental HepG2 cells were transfected with exon 3 deleted expression vector, β-catenin protein levels were not affected. Moreover, cellular morphology and population doubling time were not significantly altered.

Supporting Institution

TUBITAK

Project Number

114S207

Thanks

The author is grateful to The Scientific and Technological Research Council of Turkey (TUBITAK) for supporting this research with the grant number of 114S207.

References

  • Nollet, F., Berx, G., Molemans, F., van Roy, F. (1996) Genomic Organization of the Human β-Catenin Gene (CTNNB1). Genomics, 32, 413–424.
  • Ikeda, S., Kishida, S., Yamamoto, H., et al. (1998) Axin, a negative regulator of the Wnt signaling pathway, forms a complex with GSK-3β and β-catenin and promotes GSK-3β-dependent phosphorylation of β-catenin. EMBO J, 17, 1371–1384.
  • Aberle, H., Bauer, A., Stappert, J., et al. (1997) β-catenin is a target for the ubiquitin–proteasome pathway. EMBO J, 16, 3797–3804.
  • Cadigan, K.M., Nusse, R. (1997) Wnt signaling: a common theme in animal development. Genes Dev, 11, 3286–3305.
  • Peifer, M. (1997) β-Catenin as Oncogene--The Smoking Gun. Science, 80- 275, 1752 LP-1752.
  • Chen, S., Guttridge, D.C., You, Z., et al. (2001) WNT-1 Signaling Inhibits Apoptosis by Activating β-Catenin/T Cell Factor–Mediated Transcription. J Cell Biol, 152, 87 LP-96.
  • You, L., He, B., Uematsu. K,, et al. (2004) Inhibition of Wnt-1 Signaling Induces Apoptosis in β-Catenin-Deficient Mesothelioma Cells. Cancer Res, 64, 3474 LP-3478.
  • de la Taille, A., Rubin, M.A., Chen, M-W., et al. (2003) β-Catenin-related Anomalies in Apoptosis-resistant and Hormone-refractory Prostate Cancer Cells. Clin Cancer Res, 9, 1801 LP-1807.
  • Merle, P., Kim, M., Herrmann, M., et al. (2005) Oncogenic role of the frizzled-7/β-catenin pathway in hepatocellular carcinoma. J Hepatol, 43, 854–862.
  • Fodde, R., Brabletz, T. (2007) Wnt/β-catenin signaling in cancer stemness and malignant behavior. Curr Opin Cell Biol, 19, 150–158.
  • O’Roak, B.J., Vives, L., Fu, W., et al. (2012) Multiplex targeted sequencing identifies recurrently mutated genes in autism spectrum disorders. Science, 338, 1619–1622.
  • Horpaopan, S., Spier, I., Zink, A.M., et al. (2015) Genome-wide CNV analysis in 221 unrelated patients and targeted high-throughput sequencing reveal novel causative candidate genes for colorectal adenomatous polyposis. Int J Cancer, 136, E578–E589.
  • Dubruc, E., Putoux, A., Labalme, A., et al. (2014) A new intellectual disability syndrome caused by CTNNB1 haploinsufficiency. Am J Med Genet Part A, 164,1571–1575.
  • Kuechler, A., Willemsen, M.H., Albrecht, B., et al. (2015) De novo mutations in beta-catenin (CTNNB1) appear to be a frequent cause of intellectual disability: expanding the mutational and clinical spectrum. Hum Genet, 134, 97–109.
  • Gao, C., Wang, Y., Broaddus, R., et al. (2017) Exon 3 mutations of CTNNB1 drive tumorigenesis: a review. Oncotarget, 9, 5492–5508.
  • Carruba, G., Cervello, M., Miceli, MD., et al. (1999) Truncated Form of β-Catenin and Reduced Expression of Wild-Type Catenins Feature HepG2 Human Liver Cancer Cells. Ann N Y Acad Sci, 886, 212–216.
  • Sambrook, J., Russell, D.W. (2006) Purification of Nucleic Acids by Extraction with Phenol:Chloroform. Cold Spring Harb Protoc, 2006:pdb.prot4455.
  • Karaosmanoğlu, O., Banerjee, S., Sivas, H. (2018) Identification of biomarkers associated with partial epithelial to mesenchymal transition in the secretome of slug over-expressing hepatocellular carcinoma cells. Cell Oncol, 41, 439–453.
  • Cox, R.T., Pai, L.M., Kirkpatrick, C., et al. (1999) Roles of the C terminus of Armadillo in Wingless signaling in Drosophila. Genetics, 153, 319–332.
  • Mo, R., Chew, T.-L., Maher, M.T., et al. (2009) The terminal region of beta-catenin promotes stability by shielding the Armadillo repeats from the axin-scaffold destruction complex. J Biol Chem, 284, 28222–28231.

HepG2 Hücrelerinin Kısa Uçlu β-catenin Kodlayan İfade Vektörüyle Transfeksiyonu

Year 2020, Volume: 7 Issue: 1, 211 - 220, 28.06.2020
https://doi.org/10.35193/bseufbd.658677

Abstract

β-katenin, Wnt sinyalleşmesinde bir efektör proteinidir. β-katenin mutasyonları; otizm, kolon kanseri, gelişimsel gecikme, zihinsel engel, nörodejenerasyon, baş, deri, ve yüz anomalileri gibi çok sayıda hastalıkların gelişmesinde raporlanmıştır. Bu hastalıklar, özellikle ekson 3 delesyonu aracılı β-katenin kısalmalarıyla ilişkilendirilmiştir. Bu nedenle β-katenin proteininin yabani tip ve ekson 3 delesyonlu formlarının fonksiyonlarını anlamak çok sayıda hastalığın tedavisinde ilerlemeyi sağlayabilecektir. Kontrollü deneyler kurmak için, yabani tip ve ekson 3 delesyonlu β-catenin formlarını kodlayan ve aynı organizmadan kökenlenen ekspresyon vektörlerine ihtiyaç duyulabilmektedir. HepG2 hücrelerinin β-katenin proteinleri bakımından heterozigot olduğu uzun zamandır bilindiği için, bu çalışmada, yabani tip ve ekson 3 delesyonlu ekspresyon vektörlerini HepG2 hücrelerinin toplam RNA'sından oluşturmanın değerli olabileceği düşünülmüştür. Bunun için, HepG2 hücrelerinden RNA izole edilmiştir, cDNA parçaları polimeraz zincir reaksiyonu (PZR) ile çoğaltılmıştır, ifade vektörleri oluşturularak 5’-uçlarından dizilenmiştir. BLAST analizi sonrası hem ekson 3 delesyonlu hem de yabani tip β-katenin kodlayan pcDNA3.1/CTNNB1 ifade vektörlerinin E. coli hücrelerine başarıyla klonlandığı sonucuna varılmıştır. İlginç bir şekilde, HepG2 hücreleri ekson 3 delesyonlu ifade vektörü ile transfekte edildiğinde, β-katenin protein seviyesi etkilenmemiştir. Dahası hücre morfolojisi ve populasyon ikilenme zamanı anlamlı ölçüde değişmemiştir.

Project Number

114S207

References

  • Nollet, F., Berx, G., Molemans, F., van Roy, F. (1996) Genomic Organization of the Human β-Catenin Gene (CTNNB1). Genomics, 32, 413–424.
  • Ikeda, S., Kishida, S., Yamamoto, H., et al. (1998) Axin, a negative regulator of the Wnt signaling pathway, forms a complex with GSK-3β and β-catenin and promotes GSK-3β-dependent phosphorylation of β-catenin. EMBO J, 17, 1371–1384.
  • Aberle, H., Bauer, A., Stappert, J., et al. (1997) β-catenin is a target for the ubiquitin–proteasome pathway. EMBO J, 16, 3797–3804.
  • Cadigan, K.M., Nusse, R. (1997) Wnt signaling: a common theme in animal development. Genes Dev, 11, 3286–3305.
  • Peifer, M. (1997) β-Catenin as Oncogene--The Smoking Gun. Science, 80- 275, 1752 LP-1752.
  • Chen, S., Guttridge, D.C., You, Z., et al. (2001) WNT-1 Signaling Inhibits Apoptosis by Activating β-Catenin/T Cell Factor–Mediated Transcription. J Cell Biol, 152, 87 LP-96.
  • You, L., He, B., Uematsu. K,, et al. (2004) Inhibition of Wnt-1 Signaling Induces Apoptosis in β-Catenin-Deficient Mesothelioma Cells. Cancer Res, 64, 3474 LP-3478.
  • de la Taille, A., Rubin, M.A., Chen, M-W., et al. (2003) β-Catenin-related Anomalies in Apoptosis-resistant and Hormone-refractory Prostate Cancer Cells. Clin Cancer Res, 9, 1801 LP-1807.
  • Merle, P., Kim, M., Herrmann, M., et al. (2005) Oncogenic role of the frizzled-7/β-catenin pathway in hepatocellular carcinoma. J Hepatol, 43, 854–862.
  • Fodde, R., Brabletz, T. (2007) Wnt/β-catenin signaling in cancer stemness and malignant behavior. Curr Opin Cell Biol, 19, 150–158.
  • O’Roak, B.J., Vives, L., Fu, W., et al. (2012) Multiplex targeted sequencing identifies recurrently mutated genes in autism spectrum disorders. Science, 338, 1619–1622.
  • Horpaopan, S., Spier, I., Zink, A.M., et al. (2015) Genome-wide CNV analysis in 221 unrelated patients and targeted high-throughput sequencing reveal novel causative candidate genes for colorectal adenomatous polyposis. Int J Cancer, 136, E578–E589.
  • Dubruc, E., Putoux, A., Labalme, A., et al. (2014) A new intellectual disability syndrome caused by CTNNB1 haploinsufficiency. Am J Med Genet Part A, 164,1571–1575.
  • Kuechler, A., Willemsen, M.H., Albrecht, B., et al. (2015) De novo mutations in beta-catenin (CTNNB1) appear to be a frequent cause of intellectual disability: expanding the mutational and clinical spectrum. Hum Genet, 134, 97–109.
  • Gao, C., Wang, Y., Broaddus, R., et al. (2017) Exon 3 mutations of CTNNB1 drive tumorigenesis: a review. Oncotarget, 9, 5492–5508.
  • Carruba, G., Cervello, M., Miceli, MD., et al. (1999) Truncated Form of β-Catenin and Reduced Expression of Wild-Type Catenins Feature HepG2 Human Liver Cancer Cells. Ann N Y Acad Sci, 886, 212–216.
  • Sambrook, J., Russell, D.W. (2006) Purification of Nucleic Acids by Extraction with Phenol:Chloroform. Cold Spring Harb Protoc, 2006:pdb.prot4455.
  • Karaosmanoğlu, O., Banerjee, S., Sivas, H. (2018) Identification of biomarkers associated with partial epithelial to mesenchymal transition in the secretome of slug over-expressing hepatocellular carcinoma cells. Cell Oncol, 41, 439–453.
  • Cox, R.T., Pai, L.M., Kirkpatrick, C., et al. (1999) Roles of the C terminus of Armadillo in Wingless signaling in Drosophila. Genetics, 153, 319–332.
  • Mo, R., Chew, T.-L., Maher, M.T., et al. (2009) The terminal region of beta-catenin promotes stability by shielding the Armadillo repeats from the axin-scaffold destruction complex. J Biol Chem, 284, 28222–28231.
There are 20 citations in total.

Details

Primary Language English
Journal Section Articles
Authors

Oğuzhan Karaosmanoğlu 0000-0003-2028-7339

Project Number 114S207
Publication Date June 28, 2020
Submission Date December 12, 2019
Acceptance Date May 16, 2020
Published in Issue Year 2020 Volume: 7 Issue: 1

Cite

APA Karaosmanoğlu, O. (2020). The Transfection of HepG2 cells with Truncated β-Catenin Coding Expression Vector. Bilecik Şeyh Edebali Üniversitesi Fen Bilimleri Dergisi, 7(1), 211-220. https://doi.org/10.35193/bseufbd.658677